Insertional mutagenesis has been a valuable toot for creating and subsequently cloning genes for which no gene product is known. Agrobacterium T-DNA insertion has been successfully used as a mutagen in dicot plant species. However, T-DNA insertion is irreversible, excluding the use of T-DNA tagging systems to generate phenotypic revertants or new mutant alleles following excision and re-insertion. Furthermore, most cereals are not yet routinely transformable with Agrobacterium. DNA elements, able to insert at random in chromosomal DNA, e.g., transposons, are effective insertional mutagens.
Transposable elements (TEs), or transposons, first discovered in maize, are mobile genetic factors that move around the genome. The preference of certain TEs to move to linked sites (Dooner et al., 1989) makes it possible to map the initial introduced elements and use these mapped elements to generate secondary transpositions into nearby genes of interest. The insertion site and related gene can then be readily recovered using standard cloning or PCR-based procedures.
TEs can also be used for gene transfer to introduce a heterologous nucleic acid sequence into cereal plants. The advantages of such a gene delivery system are that: 1) the transgene of interest is physically moved away from the selection gene; 2) the generation of large numbers of plants with single copies of the transgene inserted in different chromosomal locations requires only two primary transformation events; and 3) the potential exists for the transgene to move to a genomic location that supports stable expression.
TEs occur in families of related sequences, defined by their ability to interact genetically. Within any one family, individual elements occur in two forms: one a structurally conserved element capable of promoting its own excision, termed the “autonomous” element, the other a structurally heterogeneous group of elements unable to promote their own excision, the so-called “non-autonomous” elements. Non-autonomous elements from one family can be trans-activated only by the autonomous member of the same family. Examples of such families of plant TEs include Activator-Dissociation (AciDs), EnhancerInhibitor/Suppressor-mutator (En/Spin) and Mutator (Mu/dMu) from maize and Transposon Antirrhinum majus (Tam) from Antirrhinurn majus. Analysis of isolated maize TE sequences revealed that they are flanked by short terminal inverted repeat sequences (cis-determinants) that are essential for transposition. Furthermore, the autonomous member of a TE family encodes a trans-acting factor (transposase) that is required for transposition.
TEs can either excise somatically, giving rise to sectors of various phenotypes in the plant body, or germinally, in either cell lineages that undergo meiosis or in the gametes themselves. Somatic excision of a TE from a gene whose phenotype can be readily visualized can result in a variegated pattern of excision-mediated gene expression, with clonal sectors of revertant cells on a mutant background. The size and shape of clonal sectors of revertant cells are determined by the developmental timing of TE excision and by the pattern of cell division within the host tissue (for a review, see Federoff, 1989). Progeny from a plant in which the TE has undergone germinal excision-mediated reversion will have a stable phenotype, ranging from null to full function depending on the “footprint” left behind by the transposon. These “footprints” result from excision-mediated deletions and/or non-template base additions. TEs themselves can undergo deletions, internal rearrangements and/or methylation-mediated inactivation converting an autonomous element into a non-autonomous element and/or altering the trans-activation pattern of non-autonomous elements.
Use of transposons to tag genes in plants was first applied to facilitate gene cloning in maize and Antirrhinurn where mutated alleles were already available and their endogenous TEs were well-characterized at both the genetic and molecular levels. For most higher plant species however, active transposons are either not available or not sufficiently characterized to be used to generate mutants or as gene delivery vehicles. Therefore, maize transposons have been used. Maize Ac was the first to be introduced successfully into a heterologous host, tobacco (Baker et al., 1986) Subsequently, the Ac-Ds system was used in other dicotyledenous species, including Arabidopsis thalliana and carrot (Van Sluys et al., 1987), potato (Knapp et al., 1988), tomato (Yoder et al., 1988), petunia (Gerats et al., 1989; Haring et al., 1989), soybean (Zhou et al., 1990), flax (Ellis et al., 1992; Lawrence et al., 1994), and lettuce (Yang et al., 1993).
The Ac-Ds transposable element system has been used in dicots to tag genes. Examples include the N viral resistance gene from tobacco (Whitham et al., 1994, U.S. Pat. No. 5,571,706), the petunia Ph6 coloration gene (Chuck et al., 1993), the tomato Cf-9 fungal resistance gene (Jones et al., 1994), the flax L6 gene for rust resistance (Lawrence et al., 1993) and developmental (Bancroft et al., 1993) and male-sterility (Aarts et al., 1993) genes from Arabidopsis. 
In addition to gene tagging, the Ac-Ds system has been proposed as a means of obtaining transgenic plants that are free of potentially problematic selectable marker genes that are typically used in transformation vectors (see U.S. Pat. Nos. 5,225,341 and 5,482,852). This strategy incorporates the transgene of interest into a Ds element, and introduces the construct either into plants that already contain an Ac-transposase gene, or co-transforms this construct with an Ac-transposase gene into the plant species of interest. Alternatively, a plant containing the Ds element including the transgene may be crossed with a plant containing the Ac-transposase gene. Subsequent transposition of the Ds element carrying the gene of interest to a site that is unlinked to the transformation vector sequences permits progeny plants carrying only the Ds element to be selected. Such progeny plants do not carry the transformation vector backbone or associated selectable markers.
Despite the reported successes of the Ac-Ds system in dicotyledenous plant species, the successful stable introduction of these elements into monocotyledenous species has been limited to rice (for a review, see Izawa et al., 1997). In that species, problems associated with too frequent excision of Ds elements in the F, generation, coupled with inactivation of the elements in later generations, have hampered efforts to use the system to tag genes (Izawa et al., 1997). Recently, a transient assay system for monitoring the activity of the Ac-Ds system in barley was reported (McElroy et al., 1997). While that report indicated that the Ac transposase was active and could cause excision of a Ds element in barley cells, the transient nature of the assay system did not address whether this transposon system could be effectively used in stable barley transformants for gene tagging or delivery. For example, it did not address whether the problems reported in stable transformants in rice might also be problematic in barley. Critically, it also did not indicate that excised Ds elements could re-integrate into the barley genome, a feature vital for effective transposon tagging or gene delivery.